High resolution DNA detection methods and devices

ABSTRACT

The present invention provides methods and devices for detecting a target nucleic acid molecule. A set of oligonucleotide probes integrated into an electric circuit, where the oligonucleotide probes are positioned such that they can not come into contact with one another, are contacted with a sample. If the sample contains a target nucleic acid molecule, one which has sequences complimentary to both probes, the target nucleic acid molecule can bridge the gap between the probes. The resulting bridge can then carry electrical current between the two probes, indicating the presence of the target nucleic acid molecule.

BACKGROUND OF THE INVENTION

DNA identification technology has numerous uses including identificationof pathogenic organisms, genetic testing, and forensics. Advances havebeen made to allow for automated screening of thousands of sequencesconcurrently. Gene chip technologies exist where DNA probes areimmobilized on a substrate such as a glass or silicon chip. A samplecontaining nucleic acid molecules is applied to the chip and the nucleicacid molecules within the sample are allowed to hybridize to the probeDNA on the chip. Fluorescence detection is typically used to identifydouble stranded nucleic acid molecule products. The advantage of thesystem is the ability to screen hundreds or thousands of sequences usingautomated systems.

Hybridization screening with fluorescence detection is a powerfultechnique for detecting nucleic acid sequences. However, in order todetect target DNA molecules, the target must first be amplified by PCRto get a reliable signal. The gene chip technology also requires asystem capable of detecting fluorescent or radioactive materials. Such asystem is expensive to use and is not amenable to a portable system forbiological sample detection and identification. Furthermore, thehybridization reactions take up to two hours. For many potential uses,such as detecting biological warfare agents, the gene chip system issimply not effective. Therefore, there is a need for a system which canrapidly detect small quantities of a target nucleic acid moleculewithout relying on PCR amplification.

SUMMARY OF THE INVENTION

The present invention provides a method for detecting a target nucleicacid molecule. A device for detecting the presence of a target nucleicacid molecule is provided having two electronic leads, where the ends ofthe leads are located near each other but are not in contact. One ormore sets of two oligonucleotide probes are attached to the electronicleads. The oligonucleotide probes are positioned such that the probescan not come into contact with one another and such that a targetnucleic acid molecule, which has two sequences complimentary to theprobes can bind to both probes concurrently. A sample which may have thetarget nucleic acid molecule is contacted with the probes underselective hybridization conditions. If the target is present it bridgesthe gap between the probes. The target nucleic acid molecule may thencarry current between the probes, or be used as a support to form aconductive wire between the two probes.

The present invention also provides a device for detecting the presenceof a target nucleic acid molecule. The device has two electronic leads,where the ends of the leads are located near each other but are not incontact. One or more sets of two oligonucleotide probes are attached tothe electronic leads. The oligonucleotide probes are positioned suchthat the probes can not come into contact with one another and such thata target nucleic acid molecule, which has two sequences complimentary tothe probes can bind to both probes concurrently.

DESCRIPTION OF THE DRAWINGS

FIG. 1 graphically depicts the method of the present invention. Twoleads are provided each having a probe which is complimentary tosequences on a target nucleic acid molecule (FIG. 1A). A target nucleicacid molecule binds to the two probes at the complimentary sequences(FIG. 1B). The complimentary strand is filled in (FIG. 1C). Nucleasesare used to remove the free ends of the target nucleic acid molecule(FIG. 1D). Current can be passed through the double stranded molecule orthe target nucleic acid molecule and probes may be coated with aconductor and then tested for current flow.

FIG. 2 is a variation on the method shown in FIG. 1 using a ligasemethod to distinguish a single base variation. The variation isidentified by the asterisk. After step D, a ligase is used. Only thosetargets which have an exact match at the ends of the probes will ligate.After ligation, the sample is heated to remove non-ligated targetmolecules (FIG. 2E). The structure in FIG. 2E is stable at highertemperatures, whereas the un-ligated structure in FIG. 2D would denatureunder heat treatment.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides devices and methods for rapidly detectingthe presence of nucleic acid molecules. The target nucleic acid moleculeeither itself, or as a support, is used to complete a electricalcircuit. The presence of the target nucleic acid molecule is indicatedby the ability to conduct an electrical signal through the circuit. Inthe case where the target nucleic acid molecule is not present, thecircuit is not be completed. Thus, the target nucleic acid molecule actsas a switch. The presence of the nucleic acid molecule provides an onsignal for an electrical circuit, whereas the lack of the targetnucleotide is interpreted as an off signal. Due to the direct detectionof the target nucleic acid molecule, the method allows for extremelysensitive detection of target molecules connect two wires.

The detection device is constructed on a support. Examples of usefulsubstrate materials include, e.g., glass, quartz and silicon as well aspolymeric substrates, e.g. plastics. In the case of conductive orsemi-conductive substrates, it will generally be desirable to include aninsulating layer on the substrate. However, any solid support which hasa non-conductive surface may be used to construct the device. Thesupport surface need not be flat. In fact, the support may be on thewalls of a chamber in a chip.

Two leads are provided having ends located close together, within thespanning distance of a target nucleic acid molecule, but not contactingone another. Current can not flow effectively between the leads withoutthe presence of a target nucleic acid molecule to bridge the two leads.Two probes specific to the target nucleic acid molecule are used. Thefirst is attached to one lead, the second to the other lead. The twoprobes are specific to sequences on the target molecule which areseparated by sufficient distance to span the region between the leads.Typically, the gap will by in micron or fractions of microns in length.However, as chip manufacturing has improved, it has become possible toshrink the distance between elements on a chip, requireing shorterlengths of target nucleic acid molecules.

The target nucleic acid molecule is passed over the two leads. If atarget molecule has a sequence complimentary to one of the probes, itcan bind to that probe. Once bound to that probe, the nucleic acidmolecule is tethered at that site. The sequence complimentary to thesecond probe can then bind to the second probe. To facilitate such areaction, the two complimentary sequences should be chosen such that thelength of the molecule in between can span the distance between the twoleads and provide flexibility for the nucleic acid molecule to moveeasily, such that the second complimentary sequence readily binds to thesecond probe.

In a preferred embodiment, the probes are selected to bind with thetarget such that they have approximately the same melting temperature.This can be done by varying the lengths of the hybridization region. A-Trich regions may have longer target sequences, whereas G-C rich regionswould have shorter target sequences.

Hybridization assays on substrate-bound oligonucleotide arrays involve ahybridization step and a detection step. In the hybridization step, ahybridization mixture containing the target and an isostabilizing agent,denaturing agent or renaturation accelerant is brought into contact withthe probes of the array and incubated at a temperature and for a timeappropriate to allow hybridization between the target and anycomplementary probes. Usually, unbound target molecules are then removedfrom the array by washing with a wash mixture that does not contain thetarget, such as hybridization buffer. This leaves only bound targetmolecules. In the detection step, the probes to which the target hashybridized are identified. In the present method the detection iscarried out by detecting a completed electronic circuit. Since thenucleotide sequence of the probes at each feature is known, identifyingthe locations at which target has bound provides information about theparticular sequences of these probes.

Including a hybridization optimizing agent in the hybridization mixturesignificantly improves signal discrimination between perfectly matchedtargets and single-base mismatches. As used herein, the term“hybridization optimizing agent” refers to a composition that decreaseshybridization between mismatched nucleic acid molecules, i.e., nucleicacid molecules whose sequences are not exactly complementary.

An isostabilizing agent is a composition that reduces the base-paircomposition dependence of DNA thermal melting transitions. Moreparticularly, the term refers to compounds that, in properconcentration, result in a differential melting temperature of no morethan about 1° C. for double stranded DNA oligonucleotides composed of ATor GC, respectively. Isostabilizing agents preferably are used at aconcentration between 1 M and 10 M, between 2 M and 6 M, between 4 M and6 M, between 4 M and 10 M and, optimally, at about 5 M. For example, 5 Magent in 2×SSPE is suitable. Betaines and lower tetraalkyl ammoniumsalts are examples of isostabilizing agents. In one embodiment, theisostabilizing agent is not an alkylammonium ion.

Betaine (N,N,N,-trimethylglycine; (Rees et al., Biochem., ( 1993 )32:137 -144), which is hereby incorporated by reference) can eliminatethe base pair composition dependence of DNA thermal stability. UnlikeTMAC1, betaine is zwitterionic at neutral pH and does not alter thepolyelectrolyte behavior of nucleic acids while it does alter thecomposition-dependent stability of nucleic acids. Inclusion of betaineat about 5 M can lower the average hybridization signal, but increasesthe discrimination between matched and mismatched probes.

A denaturing agent is a compositions that lowers the melting temperatureof double stranded nucleic acid molecules by interfering with hydrogenbonding between bases in a double-stranded nucleic acid or the hydrationof nucleic acid molecules. Denaturing agents can be included inhybridization buffers at concentrations of about 1 M to about 6 M and,preferably, about 3 M to about 5.5 M.

Denaturing agents include formamide, formaldehyde, DMSO(“dimethylsulfoxide”), tetraethyl acetate, urea, GuSCN, glycerol andchaotropic salts. As used herein, the term “chaotropic salt” refers tosalts that function to disrupt van der Waal's attractions between atomsin nucleic acid molecules. Chaotropic salts include, for example, sodiumtrifluoroacetate, sodium tricholoroacetate, sodium perchlorate,guanidine thiocyanate (“GuSCN”), and potassium thiocyanate.

A renaturation accelerant is a compound that increases the speed ofrenaturation of nucleic acids by at least 100-fold. They generally haverelatively unstructured polymeric domains that weakly associate withnucleic acid molecules. Accelerants include heterogenous nuclearribonucleoprotein (“hnRP”) A1 and cationic detergents such as,preferably, CTAB (“cetyltrimethylammonium bromide”) and DTAB (“dodecyltrimethylammonium bromide”), and, also, polylysine, spermine,spermidine, single stranded binding protein (“SSB”), phage T4 gene 32protein and a mixture of ammonium acetate and ethanol. Renaturationaccelerants can be included in hybridization mixtures at concentrationsof about 1 mu M to about 10 mM and, preferably, 1 mu M to about 1 mM.The CTAB buffers work well at concentrations as low as 0.1 mM.

Homologous nucleotide sequences can be detected by selectivelyhybridizing to each other. Selectively hybridizing is used herein tomean hybridization of DNA or RNA probes from one sequence to the“homologous” sequence under stringent or non-stringent conditions(Ausubel, et al., Eds., 1989, Current Protocols in Molecular Biology,Vol. I, Greene Publishing Associates, Inc. and John Wiley & Sons, Inc.,New York, at page 2.10.3, which is hereby incorporated by reference).Hybridization and wash conditions are also exemplified in Sambrook, etal., Molecular Cloning: A Laboratory Manual, Second Edition, Cold SpringHarbor, N.Y., (1989), which is hereby incorporated by reference.

A variety of hybridization buffers are useful for the hybridizationassays of the invention. Addition of small amounts of ionic detergents(such as N-lauroyl-sarkosine) are useful. LiCl is preferred to NaCl.Hybridization can be at 20°-65° C., usually 37° C. to 45° C. for probesof about 14 nucleotides. Additional examples of hybridization conditionsare provided in several sources, including: Sambrook et al., MolecularCloning: A Laboratory Manual (1989), 2nd Ed., Cold Spring Harbor, N.Y.;and Berger and Kimmel, “Guide to Molecular Cloning Techniques,” Methodsin Enzymology, (1987), Volume 152, Academic Press, Inc., San Diego,Calif.; Young and Davis, Proc. Natl. Acad. Sci. USA, 80:1194 (1983),which are hereby incorporated by reference.

In addition to aqueous buffers, non-aqueous buffers may also be used. Inparticular non-aqueous buffers which facilitate hybridization but havelow electrical conductivity are preferred.

The hybridization mixture is placed in contact with the array andincubated. Contact can take place in any suitable container, forexample, a dish or a cell specially designed to hold the probe array andto allow introduction of the fluid into and removal of it from the cellso as to contact the array. Generally, incubation will be attemperatures normally used for hybridization of nucleic acids, forexample, between about 20° C. and about 75° C., e.g., about 25° C.,about 30° C., about 35° C., about 40° C., about 45° C., about 50° C.,about 55° C., about 60° C. or about 65° C. For probes longer than about14 nucleotides, 37° C.-45° C. is preferred. For shorter probes, 55°C.-65° C. is preferred. More specific hybridization conditions can becalculated using formulas for determining the melting point of thehybridized region. Preferably, hybridization is carried out at atemperature at or between ten degrees below the melting temperature andthe melting temperature. More preferred, the hybridization is carriedout at a temperature at or between five degrees below the meltingtemperature and the melting temperature. The target is incubated withthe probe array for a time sufficient to allow the desired level ofhybridization between the target and any complementary probes in thearray. After incubation with the hybridization mixture, the arrayusually is washed with the hybridization buffer, which also can includethe hybridization optimizing agent. These agents can be included in thesame range of amounts as for the hybridization step, or they can beeliminated altogether. Then the array can be examined to identify theprobes to which the target has hybridized.

The target polynucleotide whose sequence is to be determined is usuallyisolated from a tissue sample. If the target is genomic, the sample maybe from any tissue (except exclusively red blood cells). For example,whole blood, peripheral blood lymphocytes or PBMC, skin, hair or semenare convenient sources of clinical samples. These sources are alsosuitable if the target is RNA. Blood and other body fluids are also aconvenient source for isolating viral nucleic acids. If the target ismRNA, the sample is obtained from a tissue in which the mRNA isexpressed. If the polynucleotide in the sample is RNA, it may be reversetranscribed to DNA, but in this method need not be converted to DNA.

Various methods exist for attaching the probes to the electroniccircuit. For example, U.S. Pat. Nos. 5,861,242; 5,861,242; 5,856,174;5,856,101; and 5,837,832, which are hereby incorporated by reference,disclose a method where light is shone through a mask to activatefunctional (for oligonucleotides, typically an —OH) groups protectedwith a photo-removable protecting group on a surface of a solid support.After light activation, a nucleoside building block, itself protectedwith a photo-removable protecting group (at the 5′-OH), is coupled tothe activated areas of the support. The process can be repeated, usingdifferent masks or mask orientations and building blocks, to placeprobes on a substrate.

Alternatively, new methods for the combinatorial chemical synthesis ofpeptide, polycarbamate, and oligonucleotide arrays have recently beenreported (see Fodor et al., Science, 251:767-773 (1991); Cho et al.,Science, 261:1303-1305 (1993); and Southern et al., Genomics13:1008-10017 (1992), which are hereby incorporated by reference). Thesearrays, or biological chips (see Fodor et al., Nature, 364:555-556(1993), which is hereby incorporated herein by reference), harborspecific chemical compounds at precise locations in a high-density,information rich format, and are a powerful tool for the study ofbiological recognition processes.

Preferably, the probes are attached to the leads through spatiallydirected oligonucleotide synthesis. Spatially directed oligonucleotidesynthesis may be carried out by any method of directing the synthesis ofan oligonucleotide to a specific location on a substrate. Methods forspatially directed oligonucleotide synthesis include, withoutlimitation, light-directed oligonucleotide synthesis, microlithography,application by ink jet, microchannel deposition to specific locationsand sequestration with physical barriers. In general these methodsinvolve generating active sites, usually by removing protective groups;and coupling to the active site a nucleotide which, itself, optionallyhas a protected active site if further nucleotide coupling is desired.

In one embodiment the lead-bound oligonucleotides are synthesized atspecific locations by light-directed oligonucleotide synthesis which isdisclosed in U.S. Pat. No. 5,143,854; PCT application WO 92/10092; andPCT application WO 90/15070. In a basic strategy of this process, thesurface of a solid support modified with linkers and photolabileprotecting groups is illuminated through a photolithographic mask,yielding reactive hydroxyl groups in the illuminated regions. A3′-O-phosphoramidite-activated deoxynucleoside (protected at the5′-hydroxyl with a photolabile group) is then presented to the surfaceand coupling occurs at sites that were exposed to light. Following theoptional capping of unreacted active sites and oxidation, the substrateis rinsed and the surface is illuminated through a second mask, toexpose additional hydroxyl groups for coupling to the linker. A second5′-protected, 3′-O-phosphoramidite-activated deoxynucleoside (C—X) ispresented to the surface. The selective photodeprotection and couplingcycles are repeated until the desired set of probes are obtained.Photolabile groups are then optionally removed and the sequence is,thereafter, optionally capped. Side chain protective groups, if present,are also removed. Since photolithography is used, the process can beminiaturized to specifically target leads in high densities on thesupport.

This general process can be modified. For example, the nucleotides canbe natural nucleotides, chemically modified nucleotides or nucleotideanalogs, as long as they have activated hydroxyl groups compatible withthe linking chemistry. The protective groups can, themselves, bephotolabile. Alternatively, the protective groups can be labile undercertain chemical conditions, e.g., acid. In this example, the surface ofthe solid support can contain a composition that generates acids uponexposure to light. Thus, exposure of a region of the substrate to lightgenerates acids in that region that remove the protective groups in theexposed region. Also, the synthesis method can use 3′-protected5′-O-phosphoramidite-activated deoxynucleoside. In this case, theoligonucleotide is synthesized in the 5′ to 3′ direction, which resultsin a free 5′ end.

The general process of removing protective groups by exposure to light,coupling nucleotides (optionally competent for further coupling) to theexposed active sites, and optionally capping unreacted sites is referredto herein as “light-directed nucleotide coupling.”

The probe molecules can be targeted to the leads through chemical andelectrical methods. The probes may be targeted to the leads by using achemical reaction for attaching the probe or nucleotide to the leadwhich preferably binds the probe or nucleotide to the lead rather thanthe support material. Alternatively, the probe or nucleotide may betargeted to the lead by building up a charge on the lead whichelectrostatically attracts the probe or nucleotide.

Nucleases can be used to remove probes which are attached to the chip orlead in the wrong position. More particularly, a target nucleic acidmolecule may be added to the probes. Targets which bind at both ends toprobes, one end to each lead, will have no free ends and will beresistant to exonuclease digestion. However, probes which are positionedso that the target can not contact both leads will be bound only oneend, leaving the molecule subject to digestion. Thus, improperly locatedprobes can be removed while protecting the properly located probes.After the protease is removed or inactivated the target nucleic acidmolecule can be removed and the device is ready for use.

Interest has been growing in the fabrication of microfluidic devices.Typically, advances in the semiconductor manufacturing arts have beentranslated to the fabrication of micromechanical structures, e.g.,micropumps, microvalves and the like, and microfluidic devices includingminiature chambers and flow passages.

A number of researchers have attempted employ these microfabricationtechniques in the miniaturization of some of the processes involved ingenetic analysis in particular. For example, published PCT ApplicationNo. WO 94/05414, to Northrup and White, incorporated herein by referencein its entirety for all purposes, reports an integrated micro-PCRapparatus for collection and amplification of nucleic acids from aspecimen. U.S. Pat. No. 5,304,487 to Wilding et al., and U.S. Pat. No.5,296,375 to Kricka et al., discuss devices for collection and analysisof cell containing samples. Similar techniques can be used to producechips which can accept a sample, release the nucleic acid molecules andthen detect the target sequences.

Micorfluidic devices are disclosed in U.S. Pat. No. 6,046,056, which ishereby incorporated by reference. The devices includes a series ofchannels fabricated into the surface of the substrate. At least one ofthese channels will typically have very small cross sectionaldimensions, e.g., in the range of from about 0.1 μm to about 500 μm.Preferably the cross-sectional dimensions of the channels will be in therange of from about 0.1 to about 200 μm and more preferably in the rangeof from about 0.1 to about 100 μm. In particularly preferred aspects,each of the channels will have at least one cross-sectional dimension inthe range of from about 0.1 μm to about 100 μm. Although generally shownas straight channels, it will be appreciated that in order to maximizethe use of space on a substrate, serpentine, saw tooth or other channelgeometries, to incorporate effectively longer channels in shorterdistances.

Manufacturing of these microscale elements into the surface of thesubstrates may generally be carried out by any number ofmicrofabrication techniques that are well known in the art. For example,lithographic techniques may be employed in fabricating, e.g., glass,quartz or silicon substrates, using methods well known in thesemi-conductor manufacturing industries such as photolithographicetching, plasma etching or wet chemical etching. Alternatively,micromachining methods such as laser drilling, micromilling and the likemay be employed.

Similarly, for polymeric substrates, well known manufacturing techniquesmay also be used. These techniques include injection molding or stampmolding methods where large numbers of substrates may be produced using,e.g., rolling stamps to produce large sheets of microscale substrates orpolymer microcasting techniques where the substrate is polymerizedwithin a micromachined mold.

The devices will typically include an additional planar element whichoverlays the channeled substrate enclosing and fluidly sealing thevarious channels to form conduits. Attaching the planar cover elementmay be achieved by a variety of means, including, e.g., thermal bonding,adhesives or, in the case of certain substrates, e.g., glass, orsemi-rigid and non-rigid polymeric substrates, a natural adhesionbetween the two components. The planar cover element may additionally beprovided with access ports and/or reservoirs for introducing the variousfluid elements needed for a particular screen.

The device may also include reservoirs disposed and fluidly connected atthe ends of the channels. A sample channel is used to introduce the testcompounds into the device. The introduction of a number of individual,discrete volumes of compounds into the sample may be carried out by anumber of methods. For example, micropipettors may be used to introducethe test compounds into the device. In preferred aspects, anelectropipettor may be used which is fluidly connected to samplechannel. Generally, an electropipettor utilizes electroosmotic fluiddirection, to alternately sample a number of test compounds, or subjectmaterials, and spacer compounds. The pipettor then delivers individual,physically isolated samples into the sample channel for subsequentmanipulation within the device.

Alternatively, the sample channel may be individually fluidly connectedto a plurality of separate reservoirs via separate channels. Theseparate reservoirs each contain a reactant compound, such as proteinsor detergents, with additional reservoirs being provided for appropriatespacer compounds. The test compounds, reactant compounds, and/or spacercompounds are then transported from the various reservoirs into thesample channels using appropriate fluid direction schemes.

The sample collection portion of a device of the present invention,whether or not on a micro scale, generally provides for theidentification of the sample, while preventing contamination of thesample by external elements, or contamination of the environment by thesample. Generally, this is carried out by introducing a sample foranalysis, e.g., preamplified sample, tissue, blood, saliva, etc.,directly into a sample collection chamber within the device. Typically,the prevention of cross-contamination of the sample may be accomplishedby directly injecting the sample into the sample collection chamberthrough a sealable opening, e.g., an injection valve, or a septum.Generally, sealable valves are preferred to reduce any potential threatof leakage during or after sample injection. Alternatively, the devicemay be provided with a hypodermic needle integrated within the deviceand connected to the sample collection chamber, for direct acquisitionof the sample into the sample chamber. This can substantially reduce theopportunity for contamination of the sample.

In addition to the foregoing, the sample collection portion of thedevice may also include reagents and/or treatments for neutralization ofinfectious agents, stabilization of the specimen or sample, pHadjustments, and the like. Stabilization and pH adjustment treatmentsmay include, e.g., introduction of heparin to prevent clotting of bloodsamples, addition of buffering agents, addition of protease or nucleaseinhibitors, preservatives and the like. Such reagents may generally bestored within the sample collection chamber of the device or may bestored within a separately accessible chamber, wherein the reagents maybe added to or mixed with the sample upon introduction of the sampleinto the device. These reagents may be incorporated within the device ineither liquid or lyophilized form, depending upon the nature andstability of the particular reagent used.

For those embodiments where whole cells, viruses or other tissue samplesare being analyzed, it will typically be necessary to extract thenucleic acids from the cells or viruses, prior to continuing with thevarious sample preparation operations. Accordingly, following samplecollection, nucleic acids may be liberated from the collected cells,viral coat, etc., into a crude extract, followed by additionaltreatments to prepare the sample for subsequent operations, e.g.,denaturation of contaminating (DNA binding) proteins, purification,filtration, desalting, and the like.

Liberation of nucleic acids from the sample cells or viruses, anddenaturation of DNA binding proteins may generally be performed byphysical or chemical methods. For example, chemical methods generallyemploy lysing agents to disrupt the cells and extract the nucleic acidsfrom the cells, followed by treatment of the extract with chaotropicsalts such as guanidinium isothiocyanate or urea to denature anycontaminating and potentially interfering proteins. Generally, wherechemical extraction and/or denaturation methods are used, theappropriate reagents may be incorporated within the extraction chamber,a separate accessible chamber or externally introduced.

Alternatively, physical methods may be used to extract the nucleic acidsand denature DNA binding proteins. U.S. Pat. No. 5,304,487, hereinincorporated by reference, discusses the use of physical protrusionswithin microchannels or sharp edged particles within a chamber orchannel to pierce cell membranes and extract their contents. Moretraditional methods of cell extraction may also be used, e.g., employinga channel with restricted cross-sectional dimension which causes celllysis when the sample is passed through the channel with sufficient flowpressure. Alternatively, cell extraction and denaturing of contaminatingproteins may be carried out by applying an alternating electricalcurrent to the sample. More specifically, the sample of cells is flowedthrough a microtubular array while an alternating electric current isapplied across the fluid flow. A variety of other methods may beutilized within the device of the present invention to effect celllysis/extraction, including, e.g., subjecting cells to ultrasonicagitation, or forcing cells through microgeometry apertures, therebysubjecting the cells to high shear stress resulting in rupture.

Following extraction, it will often be desirable to separate the nucleicacids from other elements of the crude extract, e.g., denaturedproteins, cell membrane particles, and the like. Removal of particulatematter is generally accomplished by filtration, flocculation or thelike. A variety of filter types may be readily incorporated into thedevice. Further, where chemical denaturing methods are used, it may bedesirable to desalt the sample prior to proceeding to the next step.Desalting of the sample, and isolation of the nucleic acid may generallybe carried out in a single step, e.g., by binding the nucleic acids to asolid phase and washing away the contaminating salts or performing gelfiltration chromatography on the sample. Suitable solid supports fornucleic acid binding include, e.g., diatomaceous earth, silica, or thelike. Suitable gel exclusion media is also well known in the art and iscommercially available from, e.g., Pharmacia and Sigma Chemical. Thisisolation and/or gel filtration/desalting may be carried out in anadditional chamber, or alternatively, the particular chromatographicmedia may be incorporated in a channel or fluid passage leading to asubsequent reaction chamber.

Alternatively, the interior surfaces of one or more fluid passages orchambers may themselves be derivatized to provide functional groupsappropriate for the desired purification, e.g., charged groups, affinitybinding groups and the like.

In a preferred embodiment of the invention, ligation methods may be usedto specifically identify single base differences in sequences.Previously, methods of identifying known target sequences by probeligation methods have been reported. U.S. Pat. No. 4,883,750 to N. M.Whiteley et al.; D. Y. Wu et al., Genomics, 4:560 (1989); U. Landegrenet al., Science, 241:1077 (1988); and E. Winn-Deen et al., Clin. Chem.,37:1522 (1991), which are hereby incorporated by reference. In oneapproach, known as oligonucleotide ligation assay (“OLA”), two probes orprobe elements which span a target region of interest are hybridized tothe target region. Where the probe elements basepair with adjacenttarget bases, the confronting ends of the probe elements can be joinedby ligation, e.g., by treatment with ligase. The ligated probe elementis then assayed, evidencing the presence of the target sequence.

In the present invention, one or both probes may be designed tospecifically recognize a variation in the sequence at the end of theprobe. After the target binds to the probes, the target is treated withnucleases to remove the ends of the molecules which do not bind to theprobes. The junction is then treated with ligase. If the complimentarysequence is present at the end of the probe, the ligase will ligate thetarget to the probe. The test chamber can then be heated up to denaturenon-ligated targets. Detection of the specific target can then becarried out.

In one embodiment of the invention, the probe set is contacted with atarget nucleic acid molecule and after hybridization the nucleic acidmolecules are coated with a conductor, such as a metal, as described inU.S. Patent Applications Ser. Nos. 60/095,096, 60/099,506, or Ser. No.09/315,750 which are hereby incorporated by reference. The coatednucleic acid molecule can then conduct electricity across the gapbetween the pair of probes, thus producing a detectable signalindicative of the presence of a target nucleic acid molecule.

Braun demonstrated that silver could be deposited along a DNA molecule.A three-step process is used. First, silver is selectively localized tothe DNA molecule through a Ag+/Na+ ion-exchange (Barton, in BioinorganicChemistry (eds Bertini, et al.) ch. 8 (University Science Books, MillValley, 1994, which is hereby incorporated by reference) and complexesare formed between the silver and the DNA bases (Spiro (ed.) NucleicAcid-Metal Ion Interactions (Wiley Interscience, New York 1980;Marzeilli, et al., J. Am. Chem. Soc. 99:2797 (1977); Eichorn (ed.)Inorganic Biochemistry, Vol. 2, ch 33-34 (Elsevier, Amsterdam, 1973),which are hereby incorporated by reference). The ion-exchange processmay be monitored by following the quenching of the fluorescence signalof the labeled DNA. The silver ion-exchanged DNA is then reduced to formaggregates with bound to the DNA skeleton. The silver aggregates arefurther developed using standard procedures, such as those used inphotographic chemistry (Holgate, et al., J. Histochem. Cytochem. 31:938(1983); Birell, et al., J. Histochem. Cytochem. 34:339 (1986), which arehereby incorporated by reference).

The nucleic acid molecule itself may have some conductive properties ofits own. These properties may be modified to reduce any detrimentaleffects on the function of the electronic circuit (Meade, et al, U.S.Pat. No. 5,770,369, “Nucleic Acid Mediated Electron Transfer” (1998),which is hereby incorporated by reference). Modification of theelectrical properties of the nucleic acid molecule may be made byintercalating compounds between the bases of the nucleic acid molecule,modifying the sugar-phosphate backbone, or by cleaving the nucleic acidmolecule after the circuit elements are formed. Cleavage of the nucleicacid molecule may be accomplished by irradiation, chemical treatment, orenzymatic degradation. Irradiation using gamma-radiation is preferredbecause radiation may penetrate materials coating the nucleic acidmolecule.

In another aspect of the invention, the electrical conductivity ofnucleic acid molecules is relied upon to transmit the electrical signal.Hans-Werner Fink and Christian Schoenenberger reported in Nature (1999),which is hereby incorporated by reference, that double-stranded DNAconducts electricity like a semiconductor. This flow of current can besufficient to construct a simple switch. The present invention providesan electronic detector based upon such a nucleic acid switch, which willindicate whether or not a target nucleic acid molecule is present withina sample.

Probes to the target nucleic acid molecule are immobilized within anelectrical circuit. The probes are physically located at a distancesufficient that they can not come into contact with one another. Thesample to be tested is contacted with the probes. If a nucleic acidmolecule is present in the sample which has sequences homologous orcomplementary to the two probes, the nucleic acid molecule can bridgethe gap between the probes. The detection unit can then detect anelectrical current which can flow through the nucleic acid molecule. Acomputer unit can detect the presence of the nucleic acid molecule as an“on” switch, while an unbridged probe set would be an “off” switch. Theinformation is processed by a digital computer which correlates thestatus of the switch with the presence of a particular target. Thecomputer can also analyze the results from several switches specific forthe same target, to determine specificity of binding and targetconcentration. The information could be quickly identified to the userby indicating the presence or absence of the biological material,organism, mutation, or other target of interest on the nucleic acidmolecule.

A detection device could comprise numerous different probe sets whichcould detect a wide variety of targets. Thus a detection device couldscreen for multiple target DNA molecules. For example, a detectiondevice could have probe sets directed at multiple pathogenic organisms.In that way, a sample could be screened for several pathogenssimultaneously. Each probe set would be a separate switch which wouldindicate the presence or absence of the complimentary nucleic acidmolecule.

A cell sample can be prepared by either chemical (including enzymatic)or physical disruption, or a combination thereof After lysis the samplecan be further processed. For example, the sample can be treated withRNase to remove any RNA to limit detection to DNA.

Prior to or at the point of contact with the probes, the nucleic acidmolecules in the sample are denatured. Denaturation is preferentiallycarried out by heat treatment. Denaturation can also be carried out byvarying the ionic concentration of the carrier solution or by acombination of ionic and heat treatment.

The present invention also has the advantage of being used for multiplesamples. The probe sets can be recycled by stripping the target DNAsfrom the probe sets. In a preferred embodiment the stripping isaccomplished by increasing temperature and/or salt concentration. Theprobe set is then ready for analysis of an additional sample.

The nucleic acid molecule of the present invention is preferentially aDNA or RNA molecule. In the present invention, preferred nucleic acidmolecules include RNA and DNA. RNA detection may allow for moresensitivity since RNA transcripts may be at higher levels. Also includedwithin the invention are chemically modified nucleic acid molecules ornucleic acid analogs. Such RNA or DNA analogs comprise but are notlimited to 2′-O-alkyl sugar modifications, methylphosphonate,phosphorothioate, phosphorodithioate, formacetal, 3′-thioformacetal,sulfone, sulfamate, and nitroxide backbone modifications, amides, andanalogs wherein the base moieties have been modified. In addition,analogs of oligomers may be polymers in which the sugar moiety has beenmodified or replaced by another suitable moiety, resulting in polymerswhich include, but are not limited to, polyvinyl backbones (Pitha etal., “Preparation and Properties of Poly (I-vinylcytosine),” Biochim.Biophys. Acta, 204:381-8 (1970); Pitha et al., “Poly(1-vinyluracil): ThePreparation and Interactions with Adenosine Derivatives,” Biochim.Biophys. Acta, 204:39-48 (1970), which are hereby incorporated byreference), morpholino backbones (Summerton, et al., “MorpholinoAntisense Oligomers: Design, Preparation, and Properties,” AntisenseNucleic Acid Drug Dev., 7:187-9 (1997), which is hereby incorporated byreference) and peptide nucleic acid (PNA) analogs (Stein et al., “ASpecificity Comparison of Four Antisense Types: Morpholino, 2′-O-methylRNA, DNA, and Phosphorothioate DNA,” J. Antisense Nucleic Acid DrugDev., 7:151-7 (1997); Egholm et al., “Peptide Nucleic Acids(PNA)-Oligonucleotide Analogues with an Achiral Peptide Backbone,” J.Am. Chem. Soc., 114:1895-1897 (1992); Faruqi et al., “Peptide NucleicAcid-Targeted Mutagenesis of a Chromosomal Gene in Mouse Cells,” Proc.Natl. Acad. Sci. USA, 95:1398-403 (1998); Christensen et al.,“Solid-Phase Synthesis of Peptide Nucleic Acids,” J. Pept. Sci.,1:175-83 (1995); Nielsen et al., “Peptide Nucleic Acid (PNA). A DNAMimic with a Peptide Backbone,” Bioconjug. Chem., 5:3-7 (1994), whichare hereby incorporated by reference). In addition linkages may containthe following exemplary modifications: pendant moieties, such as,proteins (including, for example, nucleases, toxins, antibodies, signalpeptides and poly-L-lysine); intercalators (e.g., acridine andpsoralen), chelators (e.g., metals, radioactive metals, boron andoxidative metals), alkylators, and other modified linkages (e.g., alphaanomeric nucleic acids). Such analogs include various combinations ofthe above-mentioned modifications involving linkage groups and/orstructural modifications of the sugar or base for the purpose ofimproving RNAseH-mediated destruction of the targeted RNA, bindingaffinity, nuclease resistance, and or target specificity.

In one embodiment, the bridging nucleic acid molecule can be made doublestranded by adding a segment of a nucleic acid molecule which iscomplimentary to the region of the target nucleic acid molecule locatedbetween the sequences complimentary to the probes. Ligase can be used toligate the fragments into one molecule. The device may be recycled bypassing through a restriction endonuclease to release the bridgingnucleic acid molecule. Alternatively, a polymerase can be used to fillin the complimentary sequence. In that case, the solution must containnucleotides for the synthesis of the complimentary strand.

Each probe set consists of two probes. Each probe may consist of one ormore copies of the oligonucleotide, where all the copies for that probeattach to the circuit so that electrical current can be carried throughthe probe and to the circuit. A connection between any of theoligonucleotides in one probe with any of the oligonucleotides in theother probe of the set will complete the circuit producing an “on”signal. If the probes consist of multiple copies of the oligonucleotidesand/or if multiple probes are used, the device can be used to quantitatethe level of the target nucleic acid molecule in the sample, by thesignal strength or the number of activated switches.

The number of probes may be increased in order to determineconcentrations of the target nucleic acid molecule. For example, severalthousand repeated probes may be produced in the detection unit. Thecircuit would be able to count the number of occupied sites.Calculations could be done by the unit to determine the concentration ofthe target molecule.

The present invention can be used for numerous applications, such asdetection of pathogens. For example, samples may be isolated fromdrinking water or food and rapidly screened for infectious organisms.This invention may also be used for DNA sequencing using hybridizationtechniques. Such methods are described in U.S. Pat. No. 5,837,832, whichis hereby incorporated by reference. The present invention may be usedto screen for mutations or polymorphisms in samples isolated frompatients.

The present invention may also be used for food and water testing. Inrecent times, there have been several large recalls of tainted meatproducts. The electronic DNA detection system can be used for thein-process detection of pathogens in foods and the subsequent disposalof the contaminated materials. This could significantly improve foodsafety, prevent food borne illnesses and death, and avoid costlyrecalls. Chips with probes that can identify common food bornepathogens, such as Salmonella and E. Coli., could be designed for usewithin the food industry.

In yet another embodiment, the present invention can be used for realtime detection of biological warfare agents: With the recent concerns ofthe use of biological weapons in a theater of war and in terroristattacks, the device could be configured into a personal sensor for thecombat soldier or into a remote sensor for advanced warnings of abiological threat. The devices which can be used to specificallyidentity of the agent, can be coupled with a modem to send theinformation to another location. Mobile devices may also include aglobal positioning system to provide both location and pathogeninformation.

In yet another embodiment, the present invention may be used to identifyan individual. A series of probes, of sufficient number to distinguishindividuals with a high degree of reliability, are placed within thedevice. Various polymorphism sites are used. Preferentially, the devicecan determine the identity to a specificity of greater than one in Imillion, more preferred is a specificity of greater than one in onebillion, even more preferred is a specificity of greater than one in tenbillion.

As an example, a flow chart is provided indicating how a cell sample canbe tested for the presence of a target nucleic acid molecule:

1. Inject sample

2. Lyse cells

3. Process lysate

4. Denature nucleic acid molecules

4. Contact sample with probe sets—under stringent conditions

5. Determine whether current can travel between a probe set

6. Correlate the current signal with a positive identification of thetarget DNA

Note that not all steps are required depending upon the application. Forexample, lysis is only needed if the DNA is still within a cell.

Control probe sets can be utilized to verify that the system is workingappropriately. The probe sets can recognize sequences known to occurwith in the sample or be nucleic acid molecules which are added to thesample.

Controls are especially useful to determine the presence of sequenceshaving a polymorphism. Control nucleic acid molecules lacking thepolymorphism may be compared in a separate test. In a preferredembodiment, the control sequence is tested at the same time in aseparate chamber in the device.

The correct control sequence will hybridize to the probes at a slightlyhigher temperature. This difference can be used to differentiate thesingle base mutant from the correct sequence. The device will indicatebinding by the correct sequence at a temperature where the mutantsequence can not bind. However, at a lower temperature, both sequenceswill bind.

In yet another embodiment, the nucleotide probes on the substrate may berandomly chosen. A linker nucleic acid molecule comprising acomplimentary sequence to the substrate bound probe and a sequencecomplimentary to the target nucleic acid molecule (See FIG. 2). Thus thelinker can be used to make the probe sequence able to detect any targetnucleic acid sequence without having to modify the device itself Ratherthe linker molecule may be bound to the substrate bound nucleic acidmolecule either before or together with the sample to be tested. Ifdesired the linker may be ligated to the substrate bound probe. Thiswould allow for the reuse of the linker with multiple samples.

The present invention can be used to monitor gene expression in cells.The level of RNA is determined using multiple switches with probescomplimentary to the target RNA molecule. Samples can be taken atvarious times after a stimulus or at different stages of development.

In yet another embodiment, the present invention can be used to sequencenucleic acid molecules. Sequencing by hybridization (SBH) is mostefficiently practiced by attaching many probes to a surface to form anarray in which the identity of the probe at each site is known. Alabeled target DNA or RNA is then hybridized to the array, and thehybridization pattern is examined to determine the identity of allcomplementary probes in the array. Contrary to the teachings of theprior art, which teaches that mismatched probe/target complexes are notof interest, the present invention provides an analytical method inwhich the hybridization signal of mismatched probe/target complexesidentifies or confirms the identity of the perfectly matchedprobe/target complexes on the array.

Techniques for sequencing a nucleic acid using a probe array have beendisclosed in PCT Application No. 92/10588, which is hereby incorporatedby reference. Each probe is located at a positionally distinguishablelocation on the substrate. When the labeled target is exposed to thesubstrate, it binds at locations that contain complementary nucleotidesequences. Through knowledge of the sequence of the probes at thebinding locations, one can determine the nucleotide sequence of thetarget nucleic acid. The technique is particularly efficient when verylarge arrays of nucleic acid probes are utilized.

In a preferred embodiment, the device consists of a detection chiphaving the microfluidic structures needed to release the nucleic acidmolecules from a sample. The nucleic acid molecules are introduced intoa chamber with the detection system having the probes. The detectionswitches are connected to a processor which can analyze the results fromthe hybridization reactions. A user interface, such as a screen isprovided for the user to read the results. In addition, the device mayhave additional information in memory or accessible by modem regardingthe organism or individual from which the target nucleic acid moleculewas derived.

EXAMPLES Example 1 Preparation of a Sample to Detect Pathogens

A sample to be tested is isolated. A common sample would be a bloodsample from a patient. The sample is injected into the device. Thesample moves into a chamber where it is treated chemically, withdetergents, and enzymatically, with proteases to free nucleic acidmolecules from cells in the sample. Heat treatment is also used tofacilitate the release of the nucleic acid molecules. For that reason,proteins used in the present invention are preferably thermostable. Themixture may then pass though a filter on the chip to partially purifythe nucleic acid molecules.

Example 2 Preparation of Oligonucleotide Probe Sets

Each oligonucleotide probe set is selected so that the two probes arecomplimentary to a portion of the target nucleic acid molecule and sothat the two portions of the target nucleic acid molecule are locatedsufficiently far apart that the nucleic acid molecule can bridge the gapbetween the two probes on the device when they are both bound. Thecomplimentary sequences will be chosen such that there is someadditional length to allow the target nucleic acid molecule to movefreely when bound by one probe, so that it may access the second probe.Preferably the molecule will not be much longer than needed to easilybridge the gap. As the length of the molecule increases the chance of itlocating the second probe decreases, because the effective concentrationof the binding site on the target molecule decreases as the volume inwhich it can move increases.

Each probe set will be attached to a substrate so that they arepositioned as discussed above.

Example 3 Testing for the Presence of the Target Nucleic Acid Molecule

The probe sets will be contacted with the nucleic acid molecules. Thetest chamber has a small volume to facilitate binding of the target tothe probe. To increase the chance of binding, the sample is circulatedmultiple times through the test chamber. The sample will flow through atest chamber containing the probe sets, at a flow rate sufficiently lowto allow the target nucleic acid molecules to bind to a probe.Conditions are determined by the length and sequence of the probe.

The conditions will be set at a level where the stringency is sufficientto eliminate non-specific binding to the probes. The target nucleic acidmolecule is contacted with the probes under stringent conditions. Thestringent conditions for hybridization are by the nucleic acid, salt,and temperature. These conditions are well known in the art and maybe-altered in order to identify or detect identical or relatedpolynucleotide sequences. Numerous equivalent conditions comprisingeither low or high stringency depend on factors such as the length andnature of the sequence (DNA, RNA, base composition), nature of thetarget (DNA, RNA, base composition), milieu (in solution or immobilizedon a solid substrate), concentration of salts and other components(e.g., formamide, dextran sulfate and/or polyethylene glycol), andtemperature of the reactions. One or more factors be may be varied togenerate conditions of either low or high stringency different from, butequivalent to, the above listed conditions.

The test chamber is then rinsed with a solution to remove unboundnucleic acid molecules. A solution which is non-conducting lowers thelevel of false positives by cutting down on conductivity mediated by thebuffer.

A current is then applied at one lead while a detector looks for asignal at the other lead. A current between the two leads is indicativeof the presence of the target nucleic acid molecule.

Although preferred embodiments have been depicted and described indetail herein, it will be apparent to those skilled in the relevant artthat various modifications, additions, substitutions, and the like canbe made without departing from the spirit of the invention and these aretherefore considered to be within the scope of the invention as definedin the claims which follow.

1. A method for detecting a target nucleic acid molecule, comprising,providing a device for detecting the presence of a target nucleic acidmolecule, comprising: two electronic leads, where an end of a first leadis located near an end of a second lead but where the leads are not incontact, and one or more sets of two oligonucleotide probes attached tothe electronic leads, where the oligonucleotide probes are positionedsuch that the probes can not come into contact with one another and suchthat a target nucleic acid molecule, which has two sequences, a firstsequence complimentary to a first probe attached to the first lead and asecond sequence complimentary to a second probe attached to the secondlead, can bind to both probes concurrently; and contacting the probeswith a sample which may have the target nucleic acid molecule underselective hybridization conditions, and determining if an electricalcurrent can be carried between the probes, said electrical currentbetween the probes indicating the presence of the target nucleic acidmolecule in the sample which has sequences complimentary to the probes.2. The method according to claim 1, wherein the nucleic acid molecule isDNA.
 3. The method according to claim 1, wherein the nucleic acidmolecule is RNA.
 4. The method according to claim 1, further comprising:coating a target nucleic acid molecule which is attached to the probeswith a conductor.
 5. The method according to claim 4, wherein theconductor is silver.
 6. The method according to claim 4, wherein theconductor is gold.
 7. The method according to claim 1, furthercomprising: contacting the target nucleic acid molecule with nucleasesafter binding with the probes.
 8. The method according to claim 1,further comprising: contacting the target nucleic acid molecule withligase after binding with the probes, and heating the target nucleicacid molecule to a temperature high enough to denature a non-ligatedtarget nucleic acid molecule from the probes.
 9. The method according toclaim 1, wherein the probes are complimentary to sequences from thegenetic material of a pathogenic bacteria.
 10. The method according toclaim 1, wherein the pathogenic bacteria is a biowarfare agent.
 11. Themethod according to claim 1, wherein the pathogenic bacteria is a foodborne pathogen.
 12. The method according to claim 1, wherein the probesare complimentary to sequences from the genetic material of a virus. 13.The method according to claim 1, wherein the probes are complimentary tosequences from the genetic material of a human.
 14. The method accordingto claim 1, wherein one or both of the probes has a sequence which iscomplimentary to a sequence having a polymorphism, where the base orbases complimentary to the polymorphism are located at the end of theprobe.
 15. A device for detecting the presence of a target nucleic acidmolecule, comprising: two electronic leads, where an end of a first leadis located near an end of a second lead but where the leads are not incontact, and one or more sets of two oligonucleotide probes attached tothe electronic leads, where the oligonucleotide probes are positionedsuch that the probes can not come into contact with one another and suchthat a target nucleic acid molecule, which has two sequences, a firstsequence complimentary to a first probe attached to the first lead and asecond sequence complimentary to a second probe attached to the secondlead, can bind to both probes concurrently.
 16. The device according toclaim 15, further comprising: a chamber for treating a sample to releasenucleic acid molecules from a sample.
 17. The device according to claim15, wherein the device contains proteins for processing the sample. 18.The device according to claim 17, wherein the protein is selected fromthe group consisting of a ligase, protease, restriction endonuclease,nuclease, or nucleic acid binding protein.
 19. The device according toclaim 17, wherein the protein is a thermostable protein.
 20. The deviceaccording to claim 15, wherein the nucleic acid molecule is DNA.
 21. Thedevice according to claim 15, wherein the nucleic acid molecule is RNA.22. The device according to claim 1, further comprising: a chamberhaving a solution for coating a target nucleic acid molecule which isattached to the probes with a conductor.
 23. The device according toclaim 22, wherein the conductor is silver.
 24. The device according toclaim 22, wherein the conductor is gold.
 25. The device according toclaim 15, further comprising: a chamber containing nucleases forcontacting the target nucleic acid after binding with the probes. 26.The device according to claim 15, further comprising: heating elementsfor heating the sample.
 27. The device according to claim 15, whereinthe probes are complimentary to sequences from the genetic material of apathogenic bacteria.
 28. The device according to claim 27, wherein thepathogenic bacteria is a biowarfare agent.
 29. The device according toclaim 27, wherein the pathogenic bacteria is a food borne pathogen. 30.The device according to claim 15, wherein the probes are complimentaryto sequences from the genetic material of a virus.
 31. The deviceaccording to claim 15, wherein the probes are complimentary to sequencesfrom the genetic material of a human.
 32. The device according to claim31, wherein one or both of the probes has a sequence which iscomplimentary to a sequence having a polymorphism, where the base orbases complimentary to the polymorphism are located at the end of theprobe.